Both the ERK and phosphatidylinositol 3-kinase (PI3K) signaling pathways can protect cells from apoptosis following withdrawal of survival factors. We have previously shown that the ERK1/2 pathway acts independently of PI3K to block expression of the BH3-only protein, Bim EL , and prevent serum withdrawal-induced cell death, although the precise mechanism by which ERK reduced Bim EL levels was unclear. By comparing Bim mRNA and Bim protein, expression we now show that the rapid expression of Bim EL following serum withdrawal cannot be accounted for simply by increases in mRNA following inhibition of PI3K. In cells maintained in serum Bim EL is a phosphoprotein. We show that activation of the ERK1/2 pathway is both necessary and sufficient to promote Bim EL phosphorylation and that this leads to a substantial increase in turnover of the Bim EL protein. ERK1/2-dependent degradation of Bim EL proceeds via the proteasome pathway because it is blocked by proteasome inhibitors and is defective at the restrictive temperature in cells with a temperaturesensitive mutation in the E1 component of the ubiquitinconjugating system. Finally, co-transfection of Bim EL and FLAG-ubiquitin causes the accumulation of polyubiquitinated forms of Bim, and this requires the ERK1/2 pathway. Our findings provide new insights into the regulation of Bim and the role of the ERK pathway in cell survival.

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Bim, a "BH3-only" protein, is expressed de novo following withdrawal of serum survival factors and promotes cell death. We have shown previously that activation of the ERK1/2 pathway promotes phosphorylation of Bim EL , targeting it for degradation via the proteasome. However, the nature of the kinase responsible for Bim EL phosphorylation remained unclear. We now show that Bim EL is phosphorylated on at least three sites in response to activation of the ERK1/2 pathway. By using the peptidylprolyl isomerase, Pin1, as a probe for proline-directed phosphorylation, we show that ERK1/2-dependent phosphorylation of Bim EL occurs at (S/T)P motifs. ERK1/2 phosphorylates Bim EL , but not Bim S or Bim L , in vitro, and mutation of Ser 65 to alanine blocks the phosphorylation of Bim EL by ERK1/2 in vitro and in vivo and prevents the degradation of the protein following activation of the ERK1/2 pathway. We also find that ERK1/2, but not JNK, can physically associate with GSTBim EL , but not GST-Bim L or GST-Bim S , in vitro. ERK1/2 also binds to full-length Bim EL in vivo, and we have localized a potential ERK1/2 "docking domain" lying within a 27-amino acid stretch of the Bim EL protein. Our findings provide new insights into the post-translational regulation of Bim EL and the role of the ERK1/2 pathway in cell survival signaling.

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The BH3-only protein, Bim, exists as three splice variants (Bim S , Bim L , and Bim EL ) of differing pro-apoptotic potency. Bim EL , the least effective killer, is degraded by the proteasome in response to phosphorylation by extracellular signal-regulated kinases 1 and 2 (ERK1/2). ERK1/2-dependent phosphorylation correlates with the presence of a domain unique to the Bim EL splice variant that includes the major ERK1/2 phosphorylation site Ser 65 . However, efficient phosphorylation by ERK1/2, c-Jun N-terminal kinase, or p38 requires the presence in the substrate of a discrete kinase-docking domain as well as the phosphoacceptor site. Here we show that the region unique to Bim EL (amino acids 41-97) harbors two potential DEF-type ERK1/2 kinase-docking domains, DEF1 and DEF2. Peptide competition assays revealed that the DEF2 peptide could act autonomously to bind active ERK1/2, whereas the DEF1 peptide did not. Truncation analysis identified a minimal region, residues 80 -97, containing the DEF2 motif as sufficient for ERK1/2 binding. Mutation of key residues in the DEF2 motif abolished the interaction of ERK1/2 and Bim EL and also abolished ERK1/2-dependent phosphorylation of Bim EL in vivo, thereby stabilizing the protein and enhancing cytotoxicity. Our results identify a new physiologically relevant functional motif in Bim EL that may account for the distinct biological properties of this splice variant.The BH3-only protein Bim is a pro-apoptotic member of the Bcl-2 protein family that links stress-induced signals to the core apoptotic machinery (1, 2). Expression of the Bim gene is induced at the transcriptional level in response to withdrawal of cytokines and survival factors due to inactivation of protein kinase B (3) or the ERK1/2 pathway (4). In addition, the JNK 1 (c-Jun N-terminal kinase) pathway promotes c-Jun-dependent Bim expression in neurons following the withdrawal of nerve growth factor (5, 6). Alternative splicing of the Bim gene gives rise to the short, long, and extra-long Bim proteins (Bim S , Bim L , and Bim EL ) (7), thereby introducing additional levels of regulation that may account for their differences in pro-apoptotic potency. For example, Bim S is the most effective killer and is the simplest form, consisting largely of the pro-death BH3 domain and a C-terminal membrane-tethering domain (7). Bim L contains an additional domain through which it can interact with dynein light chain 1 (DLC1) with the result that in viable cells Bim L is sequestered at microtubules and so is a less effective killer (8). Disruption of microtubules can cause the redistribution of Bim L to the mitochondria, and this may be due to JNK-dependent phosphorylation of Bim L at sites adjacent to the DLC1-binding site (9). Bim EL also contains the DLC1-binding site but is the least effective killer, and this may be explained by the fact that Bim EL protein stability is subject to post-translational regulation. Activation of the ERK1/2 pathway promotes the proteasomal degradation of Bim EL (10), and this correlates ...

To investigate the role of thrombin in regulating apoptosis, we have used CCl39 cells, a fibroblast cell line in which thrombin-induced cell proliferation has been extensively studied. Withdrawal of serum from CCl39 cells resulted in a rapid apoptotic response that was completely prevented by the inclusion of thrombin. The protective effect of thrombin was reversed by pertussis toxin, suggesting that cell-survival signalling pathways are activated via a G(i) or G(o) heterotrimeric GTPase. Serum-withdrawal-induced death required de novo gene expression and was preceded by the rapid de novo expression of the pro-apoptotic 'BH3-only' protein Bim (Bcl-2-interacting mediator of cell death). Thrombin strongly inhibited the up-regulation of both Bim protein and Bim mRNA. The ability of thrombin to repress Bim expression, and to protect cells from apoptosis, was reversed by U0126, a MEK1/2 [MAPK (mitogen-activated protein kinase) or ERK (extracellular-signal-regulated kinase) 1/2] inhibitor, or LY294002, a phosphoinositide 3'-kinase (PI3K) inhibitor, suggesting that both the Raf-->MEK-->ERK1/2 and PI3K pathways co-operate to repress Bim and promote cell survival. A PAR1p (protease-activated receptor 1 agonist peptide) was also able to protect cells from serum-withdrawal-induced apoptosis, suggesting that thrombin acts via PAR1 to prevent apoptosis.